De novo design of a type 2 copper metalloenzyme for the production of value-added chemicals

The oxidative degradation of recalcitrant polymeric substrates, as polysaccharides, catalyzed by lytic polysaccharide monooxygenases (LPMOs, figure 1a), may represent the first step for the valorization of waste chemicals and for their reconversion into second-generation biofuels [1]. LPMOs contain type 2 copper site, composed of two histidine residues: a N-terminal histidine, donating both the N-terminal amino group and the Nδ imidazole nitrogen in a chelating fashion, while the other is bonded to the copper through the Nε imidazole nitrogen. This peculiar active site is known as Histidine Brace (figure 1b) and is the heart of the activity displayed by LPMOs [2]. Reproducing the catalytic features of these enzymes in de novo designed protein scaffolds can be important for structure-function relationship studies, but also for the development of efficient enzymes useful in waste treatment. Here we present the de novo design, synthesis, and preliminary characterization of miniLPMO (figure 1c), a de novo designed protein with a Histidine Brace copper- binding site. MiniLPMO binds copper with a Histidine Brace-like motif, as confirmed by UV-Vis spectroscopy, and stabilizes copper-oxygen complex under mild conditions. Finally, miniLPMO shows oxidase activity, similar to natural LPMOs. [1] P. Fairley, Nature 474 (2011) S2-S5. [2] C. H. Kjaergaard, M.F. Qayyum, S. D. Wong, F. Xu, G. R. Hemsworth, D. J. Walton, N. A. Young, G. J. Davies, P. H. Walton, K.S. Johansen, K. O. Hodgson, B. Hedman, and E. I. Solomon, Proc. Natl. Acad. Sci. 111 (2014) 8797-8802